Methods for treating, diagnosing and prognosing a haematological malignancy

ABSTRACT

The invention relates to a serotonin receptor (5-HTR) inhibitor selected from the group consisting of a type 1 5-HTR inhibitor and a type 2 5-HTR inhibitor for use in the prevention and/or treatment of a haematological malignancy. Additionally, the invention relates to in vitro methods for the identification or isolation of a malignant cell from a haematological malignancy or for diagnosing a haematological malignancy based on detecting the expression of type 1 5-HTR and/or type 2 5-HTR. Furthermore, the invention relates to in vitro methods for determining the prognosis, for monitoring the effect of a therapy or for designing a customized therapy in a subject suffering from a haematological malignancy based on determining the levels of type 1 5-HTR and or type 2 5-HTR.

TECHNICAL FIELD OF THE INVENTION

The invention is related to the field of treatment, diagnostic andprognostic methods.

BACKGROUND OF THE INVENTION

Haematologic or haematopoietic malignancies are cancers of the blood orbone marrow, including leukaemia and lymphoma. Leukaemia ischaracterized by the uncontrolled accumulation of blood cells, which iscategorized into four types: acute lymphocytic leukaemia (ALL), acutemyelogenous leukaemia (AML), chronic lymphocytic leukaemia (CLL), andchronic myelogenous leukaemia (CML).

Acute myeloid leukaemia (AML), also referred to as non-lymphoid,myeloblastic, granulocytic or myelocytic leukaemia, affects variouswhite blood cells including granulocytes and monocytes. Acute leukaemiais a rapidly progressing disease that results in the accumulation ofimmature, functionless cells in the marrow and blood; the marrow oftenstops producing enough normal red cells, white cells and platelets andleukaemic cells spread to the liver, spleen, lymph nodes, centralnervous system, kidneys and gonads. Consequently, an AML patient candevelop one or more symptoms of AML such as, for example, anemia,fatigue, flu-like symptoms, bone pain, loss of appetite, weight loss,bruising easily, bleeding, and susceptibility to infection.

More than one quarter of a million adults throughout the world arediagnosed annually with acute myeloid leukaemia (AML). Despiteconsiderable progress during the past 3 decades in the therapy of AML,two-thirds of young adults and 90% of older adults still die of theirdisease.

The experience from patients with haematological malignancies, andespecially patients with AML, suggests that systemic plasma/serumcytokine profiles can be useful, both as a diagnostic tool and forprognostication of patients. However, cytokines/chemokines are releasedby a wide range of cells and are involved in a wide range of biologicalprocesses; the altered levels may therefore mainly reflect the strengthand nature of the biological processes, and the optimal clinical use ofchemokine/cytokine analyses may therefore require combination withorgan-specific biomarkers. Chemokine levels are also altered by clinicalprocedures, therapeutic interventions and the general status of thepatients (Reikvam H. et al., Toxins (Basel). February 2013; 5(2):336-362). Thus, novel diagnostic and prognostic methods are necessary toestablish an optimal assessment and management for haematologicalmalignancies.

Current treatments for AML may involve chemotherapy, radiotherapy,immunotherapy, blood transfusions, and bone marrow transplants. Themainstay of initial treatment, cytosine arabinoside (ara-C) combinedwith an anthracycline, was developed nearly 40 years ago and remains theworldwide standard of care, or alternatively a combination of from threeto eight medications such as, for example, cytarabine, daunorubicin,idarubicin, thioguanine, mitoxantrone, etoposide, and methotrexate.While current chemotherapy can result in complete remissions, the longterm disease-free survival rate for leukaemias, in particular AML, islow.

Alternative treatment approaches are directed to developing less toxicand more efficacious therapies. A variety of novel chemotherapeuticagents have been evaluated in AML including topoisomerase I inhibitorssuch as topotecan and campothecin, platinum containing agents(carboplatin) and new anti-metabolites including gemcitabine,troxcitabine and clofarabine (Smith M, et al., Critical Reviews inOncology/Hematology. 2004; 50(3): 197-222). All of these agents haveactivity against leukaemic blasts but their use remains investigational.MDR-1 blockade using cyclosporine A or PSC 833 is not of proven benefitand may either increase toxicity or necessitate dose reduction and hencereduce overall chemotherapy exposure (Larson R A. et al, Leukemia 2003;17: 488-91).

Because chemotherapy also usually kills normal cells, patients receivingchemotherapy often experience side effects such as, for example, nausea,fatigue, and higher risk of infection.

Therefore, there is a clear and unmet need for effective therapeuticsfor treatment of haematologic malignancies, including leukaemias, withreduced toxicity.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a serotonin receptor (5-HTR)inhibitor selected from the group consisting of a type 1 5-HTR inhibitorand a type 2 5-HTR inhibitor for use in the prevention and/or treatmentof a haematological malignancy.

In a second aspect, the invention relates to an in vitro method for theidentification of a malignant cell from a haematological malignancy in asample selected from the group consisting of bone marrow, blood andlymph nodes, said method comprising detecting the expression of type 15-HTR and/or type 2 5-HTR in said cell.

In a third aspect, the invention relates to an in vitro method fordiagnosing a haematological malignancy in a subject which comprisesidentifying malignant cells by a method of the invention.

In a fourth aspect, the invention relates to an in vitro method for theisolation of a malignant cell from a haematological malignancy in asample selected from the group consisting of bone marrow, blood andlymph nodes, said method comprising detecting the expression of type 15-HTR and/or type 2 5-HTR in said cell and isolating said cellexpressing said 5-HTR.

In a fifth aspect, the invention relates to an in vitro method fordetermining the prognosis of a subject suffering from a haematologicalmalignancy which comprises:

-   -   (a) determining the expression level of type 1 5-HTR and/or type        2 5-HTR in cells of a sample from said subject selected from the        group consisting of bone marrow, blood and lymph nodes, and    -   (b) comparing said level with a reference value        wherein a decrease of the expression level of type 1 5-HTR        and/or type 2 5-HTR with respect to the reference value is        indicative that the subject shows a good prognosis or        wherein an increase of the expression level of type 1 5-HTR        and/or type 2 5-HTR with respect to the reference value is        indicative that the subject shows a bad prognosis.

In a sixth aspect, the invention relates to an in vitro method formonitoring the effect of a therapy in a subject suffering from ahaematological malignancy and being treated with said therapy whichcomprises:

-   -   a) determining the expression level of type 1 5-HTR and/or type        2 5-HTR in cells of a sample from said subject selected from the        group consisting of bone marrow, blood and lymph nodes, and    -   b) comparing said level with the expression level of type 1        5-HTR and/or type 2 5-HTR in cells of a sample from said subject        at an earlier point of time        wherein a decrease of the expression level of type 1 5-HTR        and/or type 2 5-HTR with respect to the level determined in a        sample from said subject at an earlier point of time is        indicative that the therapy is being effective or wherein an        increase of the expression level of type 1 5-HTR and/or type 2        5-HTR with respect to the level determined in a sample from said        subject at an earlier point of time is indicative that the        therapy is being ineffective.

In a seventh aspect, the invention relates to an in vitro method fordesigning a customized therapy for a subject diagnosed with ahaematological malignancy which comprises

-   -   a) determining the levels of the type 1 5-HTR and/or type 2        5-HTR-expressing cells in a sample from said subject selected        from the group consisting of bone marrow, blood and lymph nodes,        and    -   b) comparing said levels with a reference value        wherein increased levels of type 1 5-HTR and/or type 2        5-HTR-expressing cells with respect to the reference value are        indicative that the subject is to be treated with a type 1 5-HTR        inhibitor and/or a type 2 5-HTR inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Apomorphine treatment reduced AML cell viability. 10⁶ HL-60,KG-1, MonoMac-1 and Kasumi-1 cells per mL in complete RPMI media weretreated with 0.1, 1 and 10 μM Apomorphine for 72 h. Cells were incubatedat 37° C. and 5% CO₂. Cells were stained with 7-aminoactinomycin D(7-AAD, CAS no. 7240-37-1) and Hoechst 33342 (CAS no. 23491-52-3) andanalyzed by flow cytometry. Nuclear cells were identified by Hoechstpositive staining, while death cells were excluded by 7-AAD positivestaining. Cell number was calculated by volumetric count. A. The mean(symbol) for each AML cell line and standard deviation (error bars) from6 different biological replicates are represented. B. The averagecorresponding to the four different AML cell lines is represented. *p<0.05.

FIG. 2. Type 1 and 2 5HTR antagonists induced cell death on AML celllines. 10⁶ HL-60, KG-1, MonoMac-1 and Kasumi-1 AML cell lines per mLwere incubated with Apomorphine, Methiothepin, Amperozide or GR113808 at0.1, 1 and 10 μM for 72 h at 37° C. and 5% CO₂. Nuclear cells wereidentified by Hoechst positive staining, while death cells were excludedby 7-AAD positive staining. Cell number was calculated by volumetriccount. A. The mean (symbol) for each AML cell line and standarddeviation (error bars) from 6 different biological replicates arerepresented. B. AML cell lines were treated with 10 μM Apomorphine orMethiothepin in the presence or absence of 5-CT or 5-HT. * p<0.05. Apo:apomorphine; Methio: methiothepine.

FIG. 3. 5HTR antagonists reduced cell viability of primary patient AMLsamples at 24 h. 5×10⁶ primary AML cells per mL were treated withApomorphine or Methiothepin at 0.1, 1 and 10 μM in complete IMDM mediafor 24 h at 37° C. and 5% CO₂. AML bulk population was identified basedon their CD45 expression and SSC profile by cytometry. The primitive AMLpopulation was identified within the AML bulk population based on theirpositivity to CD34 and negativity to CD38. Nuclear cells were identifiedby Hoechst positive staining, while death cells were excluded by 7-AADpositive staining. Cell number was calculated by volumetric count. 11primary AML samples were analyzed in triplicates. Each symbol representsa specific AML patient. Bars represent the cell viability mean of eachreplicate. * p<0.05

FIG. 4. 5HTR antagonists reduced cell viability of primary patient AMLsamples at 72 h. 5×10⁶ primary AML cells per mL were treated withApomorphine or Methiothepin at 0.1, 1 and 10 μM in complete IMDM mediafor 72 h at 37° C. and 5% CO₂. AML bulk population was identified basedon their CD45 expression and SSC profile by cytometry. The primitive AMLpopulation was identified within the AML bulk population based on theirpositivity to CD34 and negativity to CD38. Nuclear cells were identifiedby Hoechst positive staining, while death cells were excluded by 7-AADpositive staining. Cell number was calculated by volumetric count. 11primary AML samples were analyzed in triplicates. Each symbol representsa specific AML patient. Bars represent the cell viability mean of eachreplicate. * p<0.05.

FIG. 5. 5HTR antagonists induced myeloid differentiation on AML celllines. 10⁶ HL-60, KG-1, MonoMac-1 and Kasumi-1 AML cells per mL weretreated with Apomorphine and Methiothepin at 0.1, 1 and 10 μM for 72 hat 37° C. and 5% CO₂ in complete RPMI media. The surface expression ofCD11c, CD11b and CD14 was detected by flow cytometry. Death cells wereexcluded by 7-AAD-positive staining. * p<0.05. MFI: Mean FluorescenceIntensity.

FIG. 6. 5HTR antagonist Apomorphine induced myeloid differentiation onprimary patient AML samples. 5×10⁶ primary AML cells per mL were treatedwith Apomorphine at 0.1, 1 and 10 μM in complete IMDM media for 72 h at37° C. and 5% CO₂. AML bulk population was identified based on theirCD45 expression and SSC profile by cytometry. The surface expression ofCD11b, CD14 and CD15 was detected by flow cytometry. Death cells wereexcluded by 7-AAD-positive staining. 9 primary AML samples were analyzedin triplicates. Each symbol represents a specific AML patient. Barsrepresent the cell viability mean of each replicate. * p<0.05.

FIG. 7. 5HTR antagonist Methiothepin induced myeloid differentiation onprimary patient AML samples. 5×10⁶ primary AML cells per mL were treatedwith Methiothepin at 0.1, 1 and 10 μM in complete IMDM media for 72 h at37° C. and 5% CO₂. AML bulk population was identified based on theirCD45 expression and SSC profile by cytometry. The surface expression ofCD11b, CD14 and CD15 was detected by flow cytometry. Death cells wereexcluded by 7-AAD-positive staining. 9 primary AML samples were analyzedin triplicates. Each symbol represents a specific AML patient. Linesrepresent the cell viability mean of each replicate. * p<0.05.

FIG. 8. Healthy blood cell viability remains unaffected after treatmentwith 5HTR antagonists. 5×10⁶ ficoll-isolated mononuclear cells fromperipheral blood samples obtained from healthy donors were treated withApomorphine or Methiothepin at 0.1, 1 and 10 μM per 6 mL in completeRPMI media for 24 h (A) or 72 h (B) at 37° C. and 5% CO₂. Blood cellswere identified based on their CD45 expression and SSC profile bycytometry. Nuclear cells were identified by Hoechst positive staining,while death cells were excluded by 7-AAD positive staining. Cell numberwas calculated by volumetric count. 4 primary donor samples wereanalyzed in triplicates. Bars represent the cell viability mean of eachreplicate and error bars indicate the standard deviation. * p<0.05.

FIG. 9. 5HTR antagonist treatment spared healthy hematopoieticstem/progenitor cells. 2×10⁵ lineage-depleted ficoll-isolatedmononuclear cells from umbilical cord blood samples obtained fromhealthy donors were treated with Apomorphine at 10 μM in complete IMDMmedia for 24 h at 37° C. and 5% CO₂. Blood cells were identified basedon their CD45 expression and SSC profile by cytometry. CD34 and CD38surface staining were performed to identify each subpopulation. Nuclearcells were identified by Hoechst positive staining, while death cellswere excluded by 7-AAD positive staining. Cell number was calculated byvolumetric count. 3 primary umbilical cord blood samples were analyzedin triplicates. Bars represent the cell viability mean of each replicateand error bars indicate the standard deviation.

FIG. 10. The clonogenic capacity of primary AML samples is reduced upon5HTR antagonist treatment. 5×10⁵ primary patient AML blood cells per mLwere treated with Apomorphine or Methiothepin at 10 μM in complete IMDMmedia for 18 h at 37° C. and 5% CO₂. Cells were then cultivated inMethylcellulose supplemented with hematopoietic cytokines for 14 days.The number of CFU-B obtained was measured by microscopy based onmorphological criteria. 7 primary AML blood samples were analyzed. Eachsymbol represents a specific patient sample. Lines represent themedian. * p<0.05. CFU-B: Blast colony-forming units.

FIG. 11. 5HTR antagonist treatment did not decrease the clonogeniccapacity of healthy haematopoietic stem cells. 1×10³ primarylineage-depleted umbilical cord blood cells per mL were treated withApomorphine or Methiothepin at 10 μM in complete IMDM media for 18 h at37° C. and 5% CO₂. Cells were then cultivated in Methylcellulosesupplemented with haematopoietic cytokines for 14 days. The number ofcolonies obtained was measured by microscopy based on morphologicalcriteria. A. Total number of colonies is represented. CFU:colony-forming units. B. Frequency of each colony subtype is shown(CFU-Mix, Mix lineaged colony; CFU-GM, granulo-monocyte colony; CFU-G,granulocyte colony; CFU-M, monocyte colony; BFU-E erythrocyte blastcolony). 4 primary umbilical cord blood samples were analyzed. Barsrepresent the cell viability mean of each replicate and error barsindicate the standard deviation. * p<0.05.

FIG. 12. AML samples differentially express 5HTRs. Primary AML bloodsamples, healthy peripheral blood samples and AML cell lines (HL-60,KG-1, MonoMac-1 and Kasumi-1) were staining on the surface for 5HTR 1Aand HTR1B. Samples were analyzed by flow cytometry and the frequency ofpositive cells is represented. 18 primary patient AML peripheral bloodsamples and 16 healthy donor peripheral blood samples are represented.Circles: AML cell lines, Squares: AML, Diamonds: healthy mature bloodcells * p<0.05; *** p<0.001.

FIG. 13. 5HTR mRNA levels correlates with AML clinical outcome. TotalRNA from primary patient AML blood samples was isolated and 5HTR1A (A)and 5HTR1B (B) mRNA levels were measured by semi-quantitative PCR.Expression level was normalized against GAPDH and calculated followingthe 2^(−ΔCt) method. 6 AML samples corresponding to the good prognosisgroup and 8 AML samples corresponding to the bad prognosis group arerepresented in triplicates. * p<0.05.

FIG. 14. Treatment with 5HTR antagonists produce a reduction insecondary CFUs. 10⁶ primary AML patient cells per mL from primary CFUspretreated with 10 apomorphine, 10 μM methiothepin or vehicle controlwere replated in methylcellulose supplemented with hematopoieticcytokines for 14 days. The number of CFU-Bs obtained was measured bymicroscopy based on morphological criteria. Three primary AML sampleswere tested. * p<0.05, *** p<0.005, **** p<0.001.

FIG. 15. Treatment with 5HTR1 antagonists reduce AML burden in an invivo xenotransplantation mouse model. 6-8 week-old NOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ (NSG) mice were myeloablated with busulfan (30 mg/kgIP) at day 0. At day 1, 10⁶ MonoMac-1 cells were injected IV. From day 7to day 14, mice were treated IP every other day with apomorphine (5mg/kg) or methiothepin (0.1 mg/kg), while control mice were treated withsaline vehicle (0.9% NaCl). At day 15, mice were sacrificed and theirbones (iliac crests, femurs and tibias) were harvested and analyzed forthe presence of human cells. Bone marrow samples were measured by flowcytometry. Frequency of human CD45 positive cells (upper panel) andtotal human CD45 positive cells (lower panel) in BM refer to control.Bars represent the mean value. Error bars represent SEM. ** p<0.01; ***p<0.001.

FIG. 16. 5HTR antagonist treatment reduces the leukemia regenerationcapacity of primary AML cells, whilst sparing healthy hematopoieticstem/progenitor cells. 1-10×10⁶ primary AML patient cells or 10-14×10⁴lineage-depleted CB cells were ex vivo treated (10 μM apomorphine, 10 μMmethiothepin or vehicle control) for 18 h in IMDM supplemented with 5%heat-inactivated fetal bovine serum and hematopoietic cytokines. Cellswere injected IV in previously busulfan (30 mg/kg)-conditioned NSG miceand left 8 weeks untreated. Mice were sacrificed and their bones (iliaccrests, femurs and tibias) were harvested and analyzed for the presenceof human leukemia (upper panel) or healthy blood (lower panel) cells byflow cytometry. Four AML patient samples and three umbilical cord blood(UCB) samples were tested. * p<0.05; ** p<0.01.

FIG. 17. Treatments with 5HTR antagonists reduce the leukemiaregeneration capacity of primary AML samples in secondary recipient,without affecting normal hematopoiesis. Upper panel. 3-10×10⁵ engraftedbone marrow cells from primary transplanted mice were injectedintravenously into secondary recipients (conditioned NSG mice) and leftuntreated for 8 weeks. Bone marrow cells were analyzed as in FIG. 16.Frequency of human CD45 positive cells referred to control isrepresented. Lower panel. 50×10³ engrafted human cells were screened forCFUs. The normalized number of CFUs obtained is represented. * p<0.05;*** p<0.005; **** p<0.0001.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have found that haematologicalmalignant cells, particularly AML cells, express serotonin receptors(5HTR) and that inhibition of type 1 and/or type 2 5HTR has cytotoxiceffect on AML cells, as shown in Examples 1 and 2, reduces theclonogenic capacity of AML blasts while sparing healthy hematopoieticstem cells (Example 5), and that type 1 and/or type 2 5-HTR inhibitorshave no effect neither on healthy blood cells nor on healthyhaematopoietic stem/progenitor cells (Example 4). Additionally, theyhave observed that the detection of the expression of type 1 5-HTRand/or type 2 5-HTR can be used for the identification of a malignantcell from a haematological malignancy (Example 6), and that a decreasein the expression of type 1 5-HTR and/or type 2 5-HTR in a sample from apatient suffering a haematological malignancy correlates with goodprognosis (Example 7).

Definitions

“Serotonin receptors”, also known as 5-hydroxytryptamine receptors or5-HT receptors or 5-HTR, as used herein, are a group of Gprotein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs)found in the central and peripheral nervous systems.

The term “type 1 5-HT receptor” or “type 1 5-HTR” or “5-HT1 receptor” or“5-HTR1”, as used herein, relates to a subfamily of 5-HT receptors thatbind the endogenous neurotransmitter serotonin (5-hydroxytryptamine,5-HT). The 5-HT1 receptor subfamily consists of five G protein-coupledreceptors (GPCRs) that are coupled to Gi/Go and the term includes5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR1E, and 5-HTR1F. These receptors mediateinhibitory neurotransmission by decreasing cellular levels of cAMP. Thecomplete protein sequence for human type 1A 5-HT receptor has theUniProt accession number P08908 (Apr. 16, 2014). The complete proteinsequence for human type 1B 5-HT receptor has the UniProt accessionnumber P28222 (Apr. 16, 2014). The complete protein sequence for humantype 1D 5-HT receptor has the UniProt accession number P28221 (Apr. 16,2014). The complete protein sequence for human type 1E 5-HT receptor hasthe UniProt accession number P28566 (May 14, 2014). The complete proteinsequence for human type 1F 5-HT receptor has the UniProt accessionnumber P30939 (Apr. 16, 2014).

The term “type 2 5-HT receptor” or “type 2 5-HTR” or “5-HT2 receptor” or“5-HTR2”, as used herein, refers to a subfamily of 5-HT receptors thatbind the endogenous neurotransmitter serotonin (5-hydroxytryptamine,5-HT). The 5-HT2 receptor subfamily consists of three G protein-coupledreceptors (GPCRs) which are coupled to Gq/G11 and the term includes5-HTR2A, 5-HTR2B, and 5-HTR2C. These receptors mediate excitatoryneurotransmission by increasing cellular levels of IP3 and DAG. Thecomplete protein sequence for human type 2A 5-HT receptor has theUniProt accession number P28223 (May 14, 2014), for human type 2B 5-HTreceptor has the UniProt accession number P41595 (May 14, 2014) and forhuman type 2C 5-HT receptor has the UniProt accession number P28335 (May14, 2014).

The term “inhibitor”, as used herein, refers to a compound inhibitingthe activity of the 5-HT receptor. The term inhibitor includes, withoutlimitation, antagonists of the 5-HT receptor, antibodies against the5-HT receptor, compounds which prevent expression of the 5-HT receptorand compounds which lead to reduced mRNA or protein levels of the 5-HTreceptor. In a preferred embodiment the inhibitor is an antagonist. Inthe context of the present invention, the term “antagonist” refers to acompound that binds to the 5-HT receptor and lacks any substantialability to activate the receptor itself. An antagonist can therebyprevent or reduce the functional activation or occupation of thereceptor by an agonist or the natural ligand when the agonist ispresent. The term “antagonist of the 5-HT receptor”, as used herein, isintended to encompass both neutral antagonists and inverse agonists. A“neutral antagonist” is a compound that blocks the action of the agonistbut has no effect on intrinsic or spontaneous receptor activity. An“inverse agonist” is able to both block the action of the agonist at thereceptor and attenuate the constitutive activity of the receptor. Theterm “antagonist” also includes competitive antagonists, which are drugsthat bind to the same site as the natural ligand; noncompetitiveantagonists which bind to a different site on the receptor than thenatural ligand; reversible antagonists which bind and unbind thereceptor at rates determined by receptor-ligand kinetics; andirreversible antagonists which bind permanently to the receptor eitherby forming a covalent bond to the active site or just by binding sotightly that the rate of dissociation is effectively zero.

The term “type 1 5-HTR inhibitor”, as used herein, refers to anycompound capable of inhibiting the activity of the 5-HTR1 (for exampleby binding to the 5-HTR1 and lacking any substantial ability to activatethe receptor itself; or by preventing or reducing the expression of5-HTR1 mRNA or 5-HTR1 protein). This term includes selective inhibitorsfor the 5-HTR1 or for any of the 5-HTR1 subtypes (5-HTR1A, 5-HTR1B,5-HTR1D, 5-HTR1E, and 5-HTR1F) and non-selective inhibitors that arealso capable of acting as inhibitors on 5-HT receptors from othersubfamilies.

The term “type 2 5-HTR inhibitor”, as used herein, refers to anycompound capable of inhibiting the activity of the 5-HTR2 (for exampleby binding to the 5-HTR2 and lacking any substantial ability to activatethe receptor itself; or by preventing or reducing the expression of5-HTR2 mRNA or 5-HTR2 protein). This term includes selective inhibitorsfor the 5-HTR2 or for any of the 5-HTR2 subtypes (5-HTR2A, 5-HTR2B and5-HTR2C) and non-selective inhibitors that are also capable of acting asinhibitors on 5-HT receptors from other subfamilies.

“Apomorphine”, as used herein, refers to(6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol,CAS number 314-19-2. Apomorphine is a type 1 and type 2 5-HTR antagonist(Millan M. J. et al. 2002. J Pharmacol Exp Ther, 303(2):791-804). In anembodiment, the inhibitor is apomorphine.

“Methiothepin” or metitepine, refers to1-methyl-4-(8-methylsulfanyl-5,6-dihydrobenzo[b][1]benzothiepin-6-yl)piperazine,CAS number 74611-28-2. Methiothepin is a type 1 and type 2 5-HTRantagonist (Kawano H. et al. 2001. Blood, 97(6):1697-1702), particularlyan inverse agonist. In an embodiment, the inhibitor is methiothepin.

“Amperozide”, as used herein, refers to4-[4,4-bis(4-fluorophenyl)butyl]-N-ethylpiperazine-1-carboxamide, CASnumber 75558-90-6. Amperozide is a type 2 5-HTR antagonist (SvartengrenJ. and Simonsson P. Pharmacol Toxicol, 1990; 66 suppl 1:8-11). In anembodiment, the inhibitor is amperozide.

The term “haematological malignancy” refers to types of cancer thataffect blood, bone marrow, and lymph nodes. Haematological malignanciesmay derive from either of the two major blood cell lineages: myeloid andlymphoid cell lines. The myeloid cell line normally producesgranulocytes, erythrocytes, thrombocytes, macrophages and mast cells;the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas,lymphocytic leukaemias, and myelomas are from the lymphoid line, whileacute and chronic myelogenous leukaemia, myelodysplastic syndromes andmyeloproliferative diseases are myeloid in origin. Non limitative,illustrative examples of haematological malignancies are Acutelymphoblastic leukaemia (ALL), Acute myelogenous leukaemia (AML),Chronic lymphocytic leukaemia (CLL), Chronic myelogenous leukaemia(CML), Acute monocytic leukaemia (AMoL), Hodgkin's lymphomas,non-Hodgkin's lymphomas and myelomas.

“Leukaemia”, as used herein, refers to a type of cancer of the blood orbone marrow characterized by an abnormal increase of immature whiteblood cells called “blasts”. Leukaemia is a broad term covering aspectrum of diseases. In turn, it is part of the even broader group ofdiseases affecting the blood, bone marrow, and lymphoid system, whichare all known as haematological neoplasms. There are four major kinds ofleukaemia: Acute lymphoblastic leukaemia, or ALL; Acute myeloidleukaemia, or AML; Chronic lymphocytic leukaemia, or CLL; Chronicmyelogenous leukaemia, or CML.

“Acute lymphoblastic leukaemia (ALL) or acute lymphoid leukaemia” is anacute form of leukaemia, or cancer of the white blood cells,characterized by the overproduction of cancerous, immature white bloodcells—known as lymphoblasts.

“Acute Myeloid Leukaemia (AML) or acute myelogenous leukaemia or acutenonlymphocytic leukaemia (ANLL)” is a cancer of the myeloid line ofblood cells, characterized by the rapid growth of abnormal white bloodcells (mieloblasts) that accumulate in the bone marrow and interferewith the production of normal blood cells. The symptoms of AML arecaused by replacement of normal bone marrow with leukaemic cells, whichcauses a drop in red blood cells, platelets and normal white bloodcells. The combination of a myeloperoxidase or Sudan black stain and anonspecific esterase stain on blood and blood marrow smears are helpfulin distinguishing AML from ALL.

“Chronic lymphocytic leukaemia (CLL) or B-cell chronic lymphocyticleukaemia (B-CLL)” is a type of cancer that causes the body to producelarge numbers of white blood cells (B cell lymphocytes).

“Chronic myelogenous leukaemia (CML)” also known as “chronicgranulocytic leukaemia (CGL)” is a type of cancer that causes the bodyto produce large numbers of white blood cells (myelocytes). In CML aproliferation of mature granulocytes (neutrophils, eosinophils andbasophils) and their precursors is found. It is associated with acharacteristic chromosomal translocation called the Philadelphiachromosome.

“Sample”, as used herein, refers to a sample selected from the groupconsisting of bone marrow, blood and lymph nodes.

“Peripheral blood”, as used herein, refers to a sample comprising thecellular components of blood, consisting of red blood cells, white bloodcells, and platelets, which are found within the circulating pool ofblood and not sequestered within the lymphatic system, spleen, liver, orbone marrow.

“Malignant cell”, as used herein, is a “tumour cell” or “cancer cell”and refers to cells that grow and divide at an unregulated, quickenedpace.

The term “subject” or “individual” or “animal” or “patient” includes anysubject, particularly a mammalian subject, for whom therapy is desired.Mammalian subjects include humans, domestic animals, farm animals, andzoo or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice,horses, cattle, cows, and so on.

“Immunocytochemistry” refers to a technique used to localize thepresence of a specific protein or antigen in cells by use of a specificprimary antibody that binds to it wherein the extracellular matrix andother stromal components are removed, leaving only whole cells to stain.

The term “decrease of the expression level” refers to the level ofexpression of type 1 5-HTR and/or type 2 5-HTR which is lower than areference value. The expression level is considered to be lower than areference value when it is at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 100%, at least 110%, at least 120%, at least 130%,at least 140%, at least 150%, or more lower than its reference value.

The term “increase of the expression level” is referred to the level ofexpression of type 1 5-HTR and/or type 2 5-HTR which is higher than areference value. The levels of expression are considered to be higherthan its reference value when they are at least 1.5%, at least 2%, atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 100%, atleast 110%, at least 120%, at least 130%, at least 140%, at least 150%,or more higher than its reference value.

The term “reference value”, as used herein, relates to a predeterminedcriteria used as a reference for evaluating the values or data obtainedfrom the samples collected from a subject. The reference value orreference level can be an absolute value; a relative value; a value thathas an upper or a lower limit; a range of values; an average value; amedian value; a mean value; or a value as compared to a particularcontrol or baseline value. A reference value can be based on anindividual sample value, such as for example, a value obtained from asample from the subject being tested, but at an earlier point in time.The reference value can be based on a large number of samples, such asfrom population of subjects of the chronological age matched group, orbased on a pool of samples including or excluding the sample to betested.

“Good prognosis”, as used herein, means an outcome which would beregarded positive for the patient and depends on the prognosis type; forexample, a good prognosis of 1 year survival (1YS) would mean that thepatient will survive for at least 1 year. In a preferred embodiment,good prognosis relates to a probability over 40% of 5 year survivalafter diagnosis of the disease.

“Bad prognosis”, as used herein, means an outcome which would beregarded negative for the patient and depends on the prognosis type; forexample, a bad prognosis of 1 year survival would mean that the patientwill not survive for at least 1 year. In a preferred embodiment, badprognosis relates to a probability under 40% of 5 year survival afterdiagnosis of the disease.

“Earlier point of time”, as used herein, refers to any moment before thetherapy is administered to a subject suffering from a haematologicalmalignancy.

“Effective therapy”, as used herein, refers to a therapy that leads toreduced levels of type 1 5-HTR and/or type 2 5-TR in a sample of thepatient suffering from a haematological malignancy and being treatedwith said therapy.

“Ineffective therapy”, as used herein, refers to a therapy that does nothelp to reduce the level of type 1 5-HTR and/or type 2 5-HTR in a sampleof the patient suffering from a haematological malignancy and beingtreated with said therapy.

1—Medical Uses

The authors of the present invention have found that type 1 5-HTR andtype 2 5-HTR inhibitors are therapeutically effective in the treatmentof a haematological malignancy, preferably AML, and that said inhibitorshave no effect neither on healthy blood cells nor on healthyhaematopoietic stem cells, thus avoiding toxicity over normal cellsproduced by classical chemotherapeutic treatments.

In a first aspect, the invention relates to a serotonin receptor (5-HTR)inhibitor selected from the group consisting of a type 1 5-HTR inhibitorand a type 2 5-HTR inhibitor for use in the prevention and/or treatmentof a haematological malignancy.

Alternatively, the invention relates to the use of a serotonin receptor(5-HTR) inhibitor selected from the group consisting of a type 1 5-HTRinhibitor and a type 2 5-HTR inhibitor for the preparation of amedicament for the prevention and/or treatment of a haematologicalmalignancy.

Alternatively, the invention relates to a method for preventing and/ortreating a haematological malignancy comprising administering aserotonin receptor (5-HTR) inhibitor selected from the group consistingof a type 1 5-HTR inhibitor and a type 2 5-HTR inhibitor to a subject inneed thereof.

The term “prevention”, “preventing” or “prevent”, as used herein,relates to the administration of an inhibitor according to the inventionor of a medicament comprising said inhibitor to a subject who has notbeen diagnosed as possibly having a haematological malignancy at thetime of administration, but who would normally be expected to developsaid disease or be at increased risk for said disease. The preventionintends to avoid the appearance of said disease. The prevention may becomplete (e.g. the total absence of a disease). The prevention may alsobe partial, such that for example the occurrence of a disease in asubject is less than that which would have occurred without theadministration of the inhibitor of the present invention. Preventionalso refers to reduced susceptibility to a clinical condition.

The term “treatment”, as used herein, relates to the administration ofan inhibitor according to the invention or of a medicament comprisingsaid inhibitor to a subject suffering from a haematological malignancyincluding the administration in an initial or early stage of a disease,wherein the object is to prevent or slow down (lessen) an undesiredphysiological change or disorder. Beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms, diminishmentof extent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. Treatment also means prolonging survival ascompared to expected survival if not receiving the treatment.

In a preferred embodiment the type 1 5-HTR inhibitor is selected fromthe group consisting of type 1A, type 1B, type 1D, type 1E and type 1F5-HTR inhibitor; preferably is type 1A 5-HTR inhibitor.

In another preferred embodiment the type 2 5-HTR inhibitor is selectedfrom the group consisting of type 2A, type 2B and type 2C 5-HTRinhibitor; preferably is selected from type 2B and type 2C 5-HTRinhibitor; more preferably is type 2C 5-HTR inhibitor.

The person skilled in the art knows how to determine the affinity of aparticular molecule for a type 1 5-HTR and/or a type 2 5-HTR and also todetermine if this particular molecule is an inhibitor of said receptor.For example, the 5-HTR affinity of a molecule can be determined usingthe methodology described by Millan et al. (Millan et al. J PharmacolExp Ther. 2002; 303(2):791-804) (radioligand binding assay) An assay toassess if a compound is a type 1 5-HTR inhibitor is the determination ofthe Gi activation status and measuring the cAMP production andactivation of adenylyl cyclase (Nichols D. E. and Nichols C. E. ChemRev, 2008; 108(5):1614-41). An assay to assess if a compound is a type 25-HTR inhibitor is the measurement of membrane phosphoinositideshydrolysis and activation of PKC (Nichols D. E. and Nichols C. E. ChemRev, 2008; 108(5):1614-41). Assays that can be performed by the personskilled in the art to distinguish if the type 1 5-HTR inhibitor is type1A, type 1B, type 1D, type 1E or type 1F 5-HTR inhibitor are competitiveactivation assays with subtype-specific agonists. Assays that can beperformed by the person skilled in the art to distinguish if the type 25-HTR inhibitor is type 2A, type 2B and type 2C 5-HTR inhibitor arecompetitive activation assays using subtype-specific agonists.

In an embodiment, the 5-HTR inhibitor is a non-selective inhibitor thatmay act as inhibitor for different types of 5-HTR. In anotherembodiment, the 5-HTR inhibitor is selective for a type 1 5-HTR and/or atype 2 5-HTR.

The type 1 or type 2 5-HTR inhibitors can be, among others, proteins,peptides, interference RNA, antisense oligonucleotides or small organicmolecules.

In a preferred embodiment, the type 1 5-HTR inhibitor and/or type 25-HTR inhibitor is selected from the compounds of Table 1 orpharmaceutically acceptable salts thereof.

TABLE 1 INHIBITORS OF TYPE 1 AND/OR TYPE 2 5- HTR FOR USE ACCORDING TOTHE INVENTION I 5-HT1A antagonists such as Methiothepin Apomorphine BMY7378 Cyanopindolol Iodocyanopindolol Lecozotan Methysergide NAN-190Nebivolol Nefazodone WAY-100,135 WAY-100,635 Mefway II 5-HT1Bantagonists such as Methiothepin Alprenolol AR-A000002 AsenapineCyanopindolol GR-127,935 Iodocyanopindolol Isamoltane MetergolineOxprenolol Pindolol Propranolol SB-216,641 Yohimbine GR-55562 SB-224289SB-236057 III 5-HT1D antagonists such as BRL-15572 GR-127,935 KetanserinMetergoline Methiothepin Rauwolscine Ritanserin Vortioxetine ZiprasidoneSB-714786 IV 5-HT2A antagonists such as Apomorphine Atypicalantipsychotics, such as Clozapine, Olanzapin, Quetiapine, Risperidone,Ziprasidone Aripiprazole Asenapine Amitriptyline ClomipramineCyproheptadine Eplivanserin Etoperidone Haloperidol HydroxyzineIloperidone Ketanserin Methysergide Mianserin Mirtazapine NefazodonePimavanserin Pizotifen Ritanserin Trazodone Yohimbine MDL-100907 V5-HT2B antagonists such as Apomorphine Agomelatine Asenapine BZPKetanserin Methysergide Ritanserin RS-127,445 Tegaserod YohimbineSB-200646 SB-204741 VI 5-HT2C antagonists such as ApomorphineAgomelatine Amitriptyline Asenapine Clomipramine ClozapineCyproheptadine Dimebolin Eltoprazine Etoperidone Fluoxetine HaloperidolIloperidone Ketanserin Lisuride Methysergide Mianserin MirtazapineNefazodone Olanzapine Paroxetine Quetiapine Risperidone RitanserinTramadol Trazodone Ziprasidone SB-242084 RS-102221 VII Other compounds,such as: (S)-UH-301 ((S)-5-fluoro-8-hydroxy-2-dipropylaminotetralin)(Moreau et al., Brain Res. Bull. 29, 901-04 (1992)) Alprenolol(1-(1-methylethyl)amino-3-[2-(2-propenyl)- phenoxy]-2-propanol)(Brandstrom et al., U.S. Pat. No. 3,466,325) Spiperone(8-[4-(4-fluorophenyl)-4-oxobutyl]-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one) (U.S. Pat. Nos. 3,155,669 and 3,155,670)Tertatolol (8-(3-t-butylamino-2-hydroxypropyloxy)- thiochroman) (U.S.Pat. No. 3,960,891) Propranolol(1-isopropylamino-3-(1-naphthalenyloxy)-2- propanol) (Crowther et al.,U.S. Pat. No. 3,337,628) Penbutolol(1-(t-butylamino)-2-hydroxy-3-(2-cyclopentyl- phenoxy)propane) (Ruschiget al., U.S. Pat. No. 3,551,493) Pindolol(4-(2-hydroxy-3-isopropylaminopropoxy)-indole), (U.S. Pat. No.3,471,515) The compounds of formula I disclosed in EP 0687472 A2 VIIIInhibitory antibodies of type 1 and/or type 2 5-HTR IX An interferenceRNA specific for the type 1 and/or type 2 5-HTR sequences X An antisenseoligonucleotide specific for the type 1 and/or type 2 5-HTR sequences XIA ribozyme or DNA enzyme specific for the type 1 and/or type 2 5-HTRsequences

In a preferred embodiment, the inhibitor is an antagonist, and morepreferred and antagonist selected from the group consisting ofapomorphine, methiothepin, amperozide and a pharmaceutically acceptablesalt thereof. The term “antagonist” has been defined previously. Theactivity of type 1 5-HTR can be determined by detecting decreasinglevels of cAMP (Williams C. Nat Rev Drug Discovery, 2004; 3(2):125-35)and increasing levels of phosphor-Aid (Suni M A. and Maino V C. MethodsMol Biol 2011; 717:155-69); and the activity of type 2 5-HTR can bedetermined by detecting increasing levels of IP3 and DAG (Thomsen W.,Frazer J. et al. Curr Opin Biotechnol, 2005; 16(6):655-65) and alsoincreasing levels of phosphor-ERK1/2 (Suni M A. and Maino V C. MethodsMol Biol 2011; 717:155-69).

The term “pharmaceutically acceptable salt thereof”, as used herein,refers to derivatives of the compounds of Table 1 wherein the parentcompound is modified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from1,2-ethanedisulfonic, 2-acetoxybenzoic, 2-hydroxyethanesulfonic, acetic,ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric,edetic, ethane disulfonic, ethane sulfonic, fumaric, glucoheptonic,gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic,hydrabamic, hydrobromic, hydrochloric, hydroiodide, hydroxymaleic,hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic,maleic, malic, mandelic, methanesulfonic, napsylic, nitric, oxalic,pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic,propionic, salicylic, stearic, subacetic, succinic, sulfamic,sulfanilic, sulfuric, tannic, tartaric, and toluenesulfonic.

The pharmaceutically acceptable salts of the compounds of Table 1 can besynthesized from the parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare useful. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 18^(th) ed., Mack Publishing Company, Easton,Pa., 1990, p. 1445.

In an embodiment, the inhibitor is an inhibitory antibody. The term“inhibitory antibody” is understood to mean, according to the presentinvention, an antibody that is capable of binding to type 1 5-HTR ortype 2 5-HTR provoking the inhibition of the activation of saidreceptors by its natural ligand. Antibodies may be prepared using anymethod known by a person skilled in the art. Thus, polyclonal antibodiesare prepared by immunization of an animal with the protein aimed to beinhibited. Monoclonal antibodies may be prepared using the methoddescribed by Kohler, Milstein et al (Nature, 1975, 256: 495). Onceantibodies capable of binding to type 1 5-HTR or type 2 5-HTR areidentified, those antibodies capable of inhibiting type 1 5-HTR or type2 5-HTR activity using the abovementioned assays for determination oftype 1 5-HTR or type 2 5-HTR activity will be selected. Suitableantibodies in the present invention include intact antibodies whichcomprise an antigen-binding variable region and a constant region,fragments “Fab”, “F(ab′)2”, “Fab′”, Fv, scFv, diabodies and bispecificantibodies.

In another embodiment, the inhibitor is an interference RNA. As usedherein, the term “interference RNA” or “iRNA” refers to RNA moleculescapable of silencing the expression of type 1 5-HTR or type 2 5-HTR geneor of any gene needed for type 1 5-HTR or type 2 5-HTR function. To thatend, iRNA are typically double-stranded oligonucleotides having at least30 base pairs in length, and they more preferably comprise about 25, 24,23, 22, 21, 20, 19, 18 or 17 ribonucleic acid base pairs. Severaldifferent types of molecules have been used effectively in iRNAtechnology including small interfering RNA (siRNA) sometimes known asshort interference RNA or silencer RNA, micro RNA (miRNA) which normallydiffer from siRNA because they are processed from single-stranded RNAprecursors and they are shown to be only partially complementary to thetarget mRNA and short hairpin RNA (shRNA).

Small interfering RNA (siRNA) agents are capable of inhibiting targetgene expression by interfering RNA. siRNAs may be chemicallysynthesized, or may be obtained by in vitro transcription, o may besynthesized in vivo in target cell. Typically, siRNAs consist of adouble-stranded RNA from 15 to 40 nucleotides in length and may containa protuberant region 3′ and/or 5′ from 1 to 6 nucleotides in length.Length of protuberant region is independent from total length of siRNAmolecule. siRNAs act by post-transcriptional degradation or silencing oftarget messenger.

siRNA may be denominated shRNA (short hairpin RNA) characterized in thatthe antiparallel strands that form siRNA are connected by a loop orhairpin region. siRNAs are constituted by a short antisense sequence (19to 25 nucleotides) followed by a loop of 5-9 nucleotides, and the sensestrand. shRNAs may be encoded by plasmids or virus, particularlyretrovirus and, more particularly, retrovirus and under the control ofpromoters such as U6 promoter for RNA polymerase III.

The siRNAs of the invention are substantially homologous to type 1 ortype 2 5-HTR mRNA or this protein-coding genome sequence. The term“substantially honomogous” is understood to mean that siRNAs have asequence sufficiently complementary or similar to target mRNA so thatsiRNA may be able to provoke mRNA degradation by RNA interference.Suitable siRNAs to provoke interference include siRNAs formed by RNA, aswell as siRNAs containing chemically different modifications such as:

-   -   siRNAs in which the links between nucleotides are different from        those appearing in nature, such as phosphorothioate links.    -   Stranded-RNA conjugates with a functional reagent, such as a        fluorophoro.    -   Modification of the ends of RNA strands, particularly the 3′ end        by the combination with different functional hydroxyl groups at        2′-position.    -   Sugar-modified nucleotides such as O-alkylated radicals at        2′-position such as 2′-O-methylribose or 2′-O-fluororibose.    -   Base-modified nucleotides such as halogenated bases (for        example, 5-bromouracil and 5-iodouracil) or alkylated bases (for        example, 7-methyl-guanosine).

The siRNAs and shRNAs of the invention may be obtained using a series oftechniques known to a person skilled in the art. For example, siRNA maybe chemically synthesized from protected ribonucleoside phosphoramiditesin a conventional DNA/RNA synthesizer. Alternatively, siRNA may beproduced by recombinant dicer from plasmid and viral vectors, where thecoding region of siRNA strand or strands is under operative control ofRNA polymerase III promoters. RNase Dicer processes shRNA into siRNA incells.

The region which is taken as a basis for the design of siRNA is notlimitative and may contain a region of coding sequence (between theinitiation codon and the termination codon) or, alternatively, maycontain sequences from the 5′ or 3′ untranslated region, preferably from25 to 50 nucleotides in length and in any position in 3′ position withregard to the initiation codon. A procedure for siRNA design involvesthe identification of sequence motive AA(N19)TT wherein N may be anynucleotide in the sequence of interest and the selection of those thatexhibit a high content in G/C. If said sequence motive is not found, itis possible to identify sequence motive NA(N21) wherein N may be anynucleotide.

In another embodiment, the inhibitor of the invention is an antisenseoligonucleotide specific to type 1 5-HTR and/or type 2 5-HTR 1, i.e.,molecules whose sequence is complementary to mRNA coding for type 15-HTR or type 2 5-HTR, i.e., complementary to cDNA coding strand. Theantisense oligonucleotide may be complementary to a complete codingregion or a region of same including both the coding region and the 5′and 3′ untranslated regions. The antisense oligonucleotides may consistof 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length.The antisense oligonucleotides may be obtained by chemical synthesis orby enzymatic binding reactions widely known to a person skilled in theart. For example, an antisense oligonucleotide may further containmodified nucleotides which increase its biological stability or thestability of the bicatenary DNA-RNA complexes formed between theantisense oligonucleotide and the target polynucleotide, such asphosphorothioate derivatives, peptide nucleic acids andacridine-substituted oligonucleotides. Modified oligonucleotides thatmay be used for the preparation of antisense nucleic acids include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetyl-citosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethyl-aminomethyl uracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcitosine, 5-methylcitosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocitosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and2,6-diaminopurine. Alternatively, the antisense nucleic acid may beproduced biologically using an expression vector in which theantisense-oriented nucleic acid has been cloned.

Another group of inhibitors that can be used in the present inventionare catalytically active nucleic acids known as ribozimes. Ribozimescomprise a catalytic region and a second region whose sequence iscomplementary to target nucleic acid and confers substrate specificityon the ribozime. After the interaction between the ribozime and itssubstrate by hybridization and coupling between complementary regions oftarget nucleic acid and ribozime, an activation of the catalytic regionis produced provoking the inter- or intramolecular rupture of targetnucleic acid. Basic considerations for the design of ribozimes arewidely known to a person skilled in the art (see, for example, Dohertyand Doudna (Annu. Rev. Biophys. Biomol. Struct. 2001; 30:457-75).

Other compounds capable of inhibiting type 1 5-HTR or type 2 5-HTRexpression that can be used in the invention include aptamers andspiegelmers. Aptamers and spiegelmers are single-stranded ordouble-stranded D- or L-nucleic acids that specifically bind to theprotein resulting in a modification of the biological activity of theprotein. Aptamers and spiegelmers are 15 to 80 nucleotides in lengthand, preferably, 20 to 50 nucleotides in length.

Suitable methods for determining whether an inhibitor is capable ofdecreasing mRNA levels include, without limitation, standard assays fordetermining mRNA expression levels such as qPCR, RT-PCR, RNA protectionanalysis, Northern blot, RNA dot blot, in situ hybridization, microarraytechnology, tag based methods such as serial analysis of gene expression(SAGE) including variants such as LongSAGE and SuperSAGE, microarrays,fluorescence in situ hybridization (FISH), including variants such asFlow-FISH, qFiSH and double fusion FISH (D-FISH), and the like.

Suitable methods for determining whether an inhibitor acts by decreasingthe type 1 or type 2 5-HTR protein levels include the quantification bymeans of conventional methods, for example, using antibodies with acapacity to specifically bind to the proteins encoded by the gene (or tofragments thereof containing antigenic determinants) and subsequentquantification of the resulting antibody-antigen complexes.

An inhibitor of the invention may inhibit type 1 or type 2 5-HTRactivity by at least 5%, at least 10%, at least 25%, at least 50%, atleast 75%, or at least 90%, and all ranges between 5% and 100%. Suitablemethods for determining whether an inhibitor acts by decreasing the type1 or type 2 5-HTR activity have been previously described.

In another preferred embodiment the inhibitor is a type 1 5-HTRinhibitor, preferably an antagonist.

According to the invention, the serotonin receptor (5-HTR) inhibitorselected from the group consisting of a type 1 5-HTR inhibitor and atype 2 5-HTR inhibitor is useful for preventing and/or treating asubject suffering a haematological malignancy. In a preferred embodimentof the invention, the subject is a mammal. In a more preferredembodiment of the invention, the subject is a human of any race and sex.In another preferred embodiment, the haematological malignancy isleukaemia, more preferred acute myeloid leukaemia (AML).

As the skilled person acknowledges, effectiveness of a type 1 5-HTRinhibitor and a type 2 5-HTR inhibitor in a haematological malignancytherapy may be demonstrated by analyzing the haematological response(measure the numbers of white cells, red cells and platelets and thelevels of hemoglobin and hematocrit), cytogenetic response and/orserological tumor markers.

Even though individual needs vary, determination of optimal ranges fortherapeutically effective amounts of the inhibitor for use according tothe invention belongs to the common experience of those experts in theart. In general, the dosage needed to provide an effective treatment,which can be adjusted by one expert in the art, will vary depending onage, health, fitness, sex, diet, weight, degree of alteration of thereceptor, frequency of treatment, nature and condition of the injury,nature and extent of impairment or illness, medical condition of thesubject, route of administration, pharmacological considerations such asactivity, efficacy, pharmacokinetic and toxicology profile of theparticular compound used, if using a system drug delivery, and if theinhibitor is administered as part of a combination of drugs.

The inhibitor of the invention may be administered by any suitableadministration route, such as, but not limited to, parenteral, oral,topical, nasal, rectal route. In a particular embodiment, the inhibitordescribed herein is administered by parenteral route, e.g. byintravenous, intrathecal, intraperitoneal, subcutaneous, intradermal,intramuscular or epidural administration.

2—Method for the Identification of a Malignant Cell from aHaematological Malignancy

The inventors of the present invention have found that malignant cellsfrom a haematological malignancy, particularly from AML, express type 15-HTR and/or type 2 5-HTR in a significantly higher quantity compared tohealthy donors. Therefore, the detection of the expression of type 15-HTR and/or type 2 5-HTR in blood cells can be useful for identifyingmalignant cells from a haematological malignancy.

In another aspect, the invention relates to an in vitro method for theidentification of a malignant cell from a haematological malignancy in asample selected from the group consisting of bone marrow, blood andlymph nodes, said method comprising detecting the expression of type 15-HTR and/or type 2 5-HTR in said cell.

As a person skilled in the art can know, the expression of type 1 5-HTRand/or type 2 5-HTR can also be detected by detecting the expression ofa functionally equivalent variant of said receptors.

“Functionally equivalent variant” is understood to mean all thoseproteins derived from type 1 5-HTR or type 2 5-HTR sequence bymodification, insertion and/or deletion or one or more amino acids,whenever the function is substantially maintained.

The activity of type 1 5-HTR can be determined by detecting decreasinglevels of cAMP and increasing levels of phospho-Akt; and the activity oftype 2 5-HTR can be determined by detecting increasing levels of IP3 andDAG and also increasing levels of phosphor-ERK1/2.

Preferably, variants of type 1 5-HTR and/or type 2 5-HTR are (i)polypeptides in which one or more amino acid residues are substituted bya preserved or non-preserved amino acid residue (preferably a preservedamino acid residue) and such substituted amino acid may be coded or notby the genetic code, (ii) polypeptides in which there is one or moremodified amino acid residues, for example, residues modified bysubstituent bonding, (iii) polypeptides resulting from alternativeprocessing of a similar mRNA, (iv) polypeptide fragments and/or (v)polypeptides resulting from type 1 5-HTR and/or type 2 5-HTR fusion orthe polypeptide defined in (i) to (iii) with another polypeptide, suchas a secretory leader sequence or a sequence being used for purification(for example, His tag) or for detection (for example, Sv5 epitope tag).The fragments include polypeptides generated through proteolytic cut(including multisite proteolysis) of an original sequence. The variantsmay be post-translationally or chemically modified. Such variants aresupposed to be apparent to those skilled in the art.

As known in the art, the “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and the substitutedamino acids preserved from a polypeptide with the sequence of a secondpolypeptide. The variants are defined to include polypeptide sequencesdifferent from the original sequence, preferably different from theoriginal sequence in less than 40% of residues per segment concerned,more preferably different from the original sequence in less than 25% ofresidues per segment concerned, more preferably different from theoriginal sequence in less than 10% of residues per segment concerned,more preferably different from the original sequence in only a fewresidues per segment concerned and, at the same time, sufficientlyhomologous to the original sequence to preserve functionality of theoriginal sequence. The present invention includes amino acid sequenceswhich are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95%similar or identical to the original amino acid sequence. The degree ofidentity between two polypeptides may be determined using computeralgorithms and methods which are widely known to those skilled in theart. The identity between two amino acid sequences is preferentiallydetermined using BLASTP algorithm [BLASTManual, Altschul, S. et al.,NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol.215: 403-410 (1990)].

The expression level of type 1 5-HTR and/or type 2 5-HTR are determinedin a sample selected from the group consisting of bone marrow, blood andlymph nodes.

A sample from a bone marrow can be obtained by aspiration and trephinebiopsy as known in the art. Blood samples can be obtained byconventional methods, using processes known in the state of the art bythe person skilled in the art, such as blood extraction by means ofpuncturing an artery or vein, normally a vein from the inner part of theelbow or from the back of the hand, the blood sample being collected inan air-tight vial or syringe. Lymph nodes are obtained by biopsy of allor part of a lymph node (excisional lymph node biopsy or incisionallymph node biopsy).

The expression “detecting the expression” refers to detecting thepresence of a haematological cell in a sample carrying a type 1 5-HTRand/or a type 2 5-HTR on its surface or expressing type 1 5-HTR and/ortype 2 5-HTR mRNA. Said detection may be qualitative or quantitative.

In an embodiment the method for identifying a malignant cell of thepresent invention does not require the determination of the level ofexpression of type 1 5-HTR and/or type 2 5-HTR.

In another embodiment, the method for identifying a malignant cell ofthe present invention comprises determining the expression level of type1 5-HTR and/or type 2 5-HTR.

The expression “determining the expression level”, as used herein,refers to determining the level of expression of a biomarker (type 15-HTR and/or type 2 5-HTR) and/or the number of cells carrying thisbiomarker on its surface (i.e. a cell surface marker). Therein, thelevel of expression refers to the level of mRNA and/or the level ofprotein and/or the number of cells carrying a biomarker on its surface.

Methods for detecting the expression can be based on detecting type 15-HTR and/or type 2 5-HTR mRNA or protein, or they also can be based ondetermining the mRNA levels or protein levels and the levels of variantsthereof, in a sample as a whole, in cells of a sample and/or in thenon-cellular fraction of a sample.

Methods for detecting mRNA are well known in the art and include, e.g.,real-time PCR (rtPCR), northern blotting, nanostring and microarraytechnologies.

By way of a non-limiting illustration, the expression levels aredetermined by means of the quantification of the levels of mRNA encodedby said genes. The latter can be quantified by means of usingconventional methods, for example, methods comprising the amplificationof mRNA and the quantification of the amplification product of saidmRNA, such as electrophoresis and staining, or alternatively, by meansof Northern blot and the use of suitable probes of the mRNA of the geneof interest or of its corresponding cDNA/cRNA, mapping with the Sinuclease, RT-PCR, hybridization, microarrays, etc. Similarly, the levelsof the cDNA/cRNA corresponding to said mRNA encoded by the marker genecan also be quantified by means of using conventional techniques; inthis event, the method of the invention includes a step of synthesis ofthe corresponding cDNA by means of reverse transcription (RT) of thecorresponding mRNA followed by the synthesis (RNA polymerase) andamplification of the cRNA complementary to said cDNA.

In order to normalize the values of mRNA expression among the differentsamples, it is possible to compare the expression levels of the mRNA ofinterest in the test samples with the expression of a control RNA. A“control RNA”, as used herein, relates to RNA whose expression levels donot change or change only in limited amounts. Preferably, the controlRNA is mRNA derived from housekeeping genes and which code for proteinswhich are constitutively expressed and carry out essential cellularfunctions. Preferred housekeeping genes for use in the present inventioninclude 18-S ribosomal protein, β-2-microglobulin, ubiquitin,cyclophilin, GAPDH, PSMB4, tubulin and β-actin.

Alternatively, it is also possible to determine the expression levels ofthe type 1 5-HTR and/or type 2 5-HTR genes by means of the determinationof the expression levels of the proteins encoded by said genes, since ifthe expression of genes is increased, an increase of the amount ofcorresponding proteins should occur and if the expression of genes isdecreased, a decrease of the amount of corresponding proteins shouldoccur.

Virtually any conventional method can be used within the frame of theinvention to detect and quantify the levels of proteins. By way of anon-limiting illustration, the expression levels are determined by meansof antibodies with the capacity for binding specifically to the proteinto be determined (or to fragments thereof containing the antigenicdeterminants) and subsequent quantification of the resultingantigen-antibody complexes. The antibodies that are going to be used inthis type of assay can be, for example, polyclonal sera, hybridomasupernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab′and F(ab′)2, scFv, diabodies, triabodies, tetrabodies and humanizedantibodies. At the same time, the antibodies may or may not be labeled.Illustrative, but non-exclusive, examples of markers that can be usedinclude radioactive isotopes, enzymes, fluorophores, chemoluminescentreagents, enzyme cofactors or substrates, enzyme inhibitors, particles,dyes, etc. There is a wide variety of well-known assays that can be usedin the present invention, using non-labeled antibodies (primaryantibody), labeled antibodies (secondary antibodies) or labeledantagonists or agonists of type 1 or 2 5-HTR; these techniques includeWestern-blot or immunoblot, ELISA (enzyme-linked immunosorbent assay),RIA (radioimmunoassay), competitive EIA (enzyme immunoassay), DAS-ELISA(double antibody sandwich ELISA), immunocytochemical andimmunohistochemical techniques, immunofluorescence, techniques based onthe use of biochips or protein microarrays including specific antibodiesor assays based on the colloidal precipitation in formats such asreagent strips. Other forms of detecting and quantifying the proteinsinclude affinity chromatography techniques, ligand-binding assays, etc.

In a preferred embodiment of the invention, the type 1 5-HTR and/or type2 5-HTR-expressing cells are detected by immunocytochemistry, preferablyby immunofluorescence.

In a preferred embodiment of the invention, detecting the expression ordetermining the levels of type 1 5-HTR and/or type 2 5-HTR is performedby immunofluorescence. Immunofluorescence (IF) is a technique used forlight microscopy with a fluorescence microscope and is used primarily onbiological samples. This technique uses the specificity of antibodies totheir antigen to target fluorescent dyes to specific biomolecule targetswithin a cell, and therefore allows visualisation of the distribution ofthe target molecule through the sample. IF is a widely used example ofimmunostaining and is a specific example of immunohisto-chemistry (IHC)or immunocytochemistry (ICC) that makes use of fluorophores to visualisethe location of the antibodies. IF can be used on tissue sections,cultured cell lines, or individual cells. IF can be used in combinationwith other, non-antibody methods of fluorescent staining, for example,use of DAPI to label DNA. Several microscope designs can be used foranalysis of IF samples; the simplest is the epifluorescence microscope,and the confocal microscope is also widely used. Varioussuper-resolution microscope designs that are capable of much higherresolution can also be used. In a preferred embodiment, theidentification of a malignant cell is performed by flow cytometry, whichis a laser-based, biophysical technology employed in cell counting, cellsorting and biomarker detection by suspending cells in a stream of fluidand passing them by an electronic detector.

According to the invention, the detection of the expression of type 15-HTR and/or type 2 5-HTR in a sample selected from the group consistingof bone marrow, blood and lymph nodes is useful for the identificationof a malignant cell from a haematological malignancy.

In a preferred embodiment, the blood sample is peripheral blood.

In another preferred embodiment, the type 1 5-HTR is 5-HTR 1A and thetype 2 5-HTR is selected from the group consisting of 5-HTR 2A, 5-HTR 2Band 5-HTR 2C. In a more preferred embodiment, the type 2 5-HTR is 5-HTR2C.

In another preferred embodiment, the method of the invention foridentifying a malignant cell comprises detecting the expression of5-HTR1A and 5-HTR2C.

In a preferred embodiment, the haematological malignancy is leukaemia,more preferably acute myeloid leukaemia.

All the terms and embodiments previously described are equallyapplicable to this aspect of the invention.

3—Method for Diagnosing a Haematological Malignancy

In another aspect, the invention relates to an vitro method fordiagnosing a haematological malignancy in a subject which comprisesidentifying malignant cells by a method according to the invention,particularly identification of a malignant cell from a haematologicalmalignancy in a sample selected from the group consisting of bonemarrow, blood and lymph nodes, said method comprising detecting theexpression of type 1 5-HTR and/or type 2 5-HTR in said cell.

Diagnosing, as used herein, refers both to the process of attempting todetermine and/or identify a possible disease in a subject, i.e. thediagnostic procedure, and to the opinion reached by this process, i.e.the diagnostic opinion. As such, it can also be regarded as an attemptat classification of an individual's condition into separate anddistinct categories that allow medical decisions about treatment andprognosis to be made. As will be understood by those skilled in the art,the diagnosis of a haematological malignancy, although preferred to be,need not be correct for 100% of the subjects to be diagnosed orevaluated. The term, however, requires that a statistically significantportion of subjects can be identified as suffering from a haematologicalmalignancy. Whether a subject is statistically significant can bedetermined without further ado by the person skilled in the art usingvarious well known statistic evaluation tools, e.g., determination ofconfidence intervals, p-value determination, Student's t-test,Mann-Whitney test, etc. Details are found in Dowdy and Wearden,Statistics for Research, John Wiley & Sons, New York 1983. Preferredconfidence intervals are at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95%. The p-values are, preferably,0.05, 0.01, 0.005 or lower.

In a preferred embodiment, the method for diagnosing a haematologicalmalignancy in a subject comprises:

-   -   (a) determining the levels of type 1 5-HTR and/or type 2        5-HTR-expressing cells in a sample from said subject selected        from the group consisting of bone marrow, blood and lymph nodes,        and    -   (b) comparing said levels with a reference value        wherein increased levels of type 1 5-HTR and/or type 2        5-HTR-expressing cells with respect to the reference value are        indicative of the subject suffering from a haematological        malignancy.

The reference value derives from a sample collection formed preferablyby a mixture of the sample to be analyzed from normal individuals notaffected by a haematological malignancy. Said reference value can bedetermined by means of techniques well known in the state of the art,for example, determining the mean of the levels of type 1 5-HTR and/ortype 2 5-HTR proteins measured in a sample taken from healthy subjects.The reference value can also be obtained from the constitutivelyexpressed proteins taken from the same subject to be analyzed.

In a preferred embodiment, the blood sample is peripheral blood. Inanother preferred embodiment, the type 1 5-HTR is 5-HTR 1A and the type2 5-HTR is selected from the group consisting of 5-HTR 2A, 5-HTR 2B and5-HTR 2C. In a more preferred embodiment, the type 2 5-HTR is 5-HTR 2C.

In another preferred embodiment, the method for diagnosing ahaematological malignancy of the invention comprises detecting theexpression of 5-HTR 1A and 5-HTR 2C.

In a preferred embodiment, the haematological malignancy is leukaemia,more preferably acute myeloid leukaemia.

In another preferred embodiment, the type 1 5-HTR and/or type 25-HTR-expressing cells are detected by immunocytochemistry, preferablyby immunofluorescence, more preferred by flow cytometry.

All the terms and embodiments previously described are equallyapplicable to this aspect of the invention.

4—Method for the Isolation of a Malignant Cell

In another aspect, the invention relates to an in vitro method for theisolation of a malignant cell from a haematological malignancy in asample selected from the group consisting of bone marrow, blood andlymph nodes, said method comprising detecting the expression of type 15-HTR and/or type 2 5-HTR in said cell and isolating said cellexpressing said 5-HTR.

The term “isolating”, as used herein, means identifying and separatingor removing a malignant cell from the remaining components present in asample selected from the group consisting of bone marrow, blood andlymph nodes.

In a preferred embodiment, the blood sample is peripheral blood.

In another preferred embodiment, the type 1 5-HTR is 5-HTR 1A and thetype 2 5-HTR is selected from the group consisting of 5-HTR 2A, 5-HTR 2Band 5-HTR 2C. In a more preferred embodiment, the type 2 5-HTR is 5-HTR2C.

In another preferred embodiment, the method of the invention forisolating a malignant cell comprises detecting the expression of 5-HTR1A and 5-HTR 2C.

In a preferred embodiment, the haematological malignancy is leukaemia,more preferably acute myeloid leukaemia.

In another preferred embodiment, the type 1 5-HTR and/or type 25-HTR-expressing cells are detected by immunocytochemistry, preferablyby immunofluorescence, more preferred by flow cytometry.

All the terms and embodiments previously described are equallyapplicable to this aspect of the invention.

5—Method for Determining the Prognosis

In another aspect, the invention relates to an in vitro method fordetermining the prognosis of a subject suffering from a haematologicalmalignancy which comprises:

-   -   (a) determining the expression level of type 1 5-HTR and/or type        2 5-HTR in cells of a sample from said subject selected from the        group consisting of bone marrow, blood and lymph nodes, and    -   (b) comparing said level with a reference value        wherein a decrease of the expression level of type 1 5-HTR        and/or type 2 5-HTR with respect to the reference value is        indicative that the subject shows a good prognosis or wherein an        increase of the expression level of type 1 5-HTR and/or type 2        5-HTR with respect to the reference value is indicative that the        subject shows a bad prognosis

In a preferred embodiment, the haematological malignancy is leukaemia,and more preferably acute myeloid leukaemia.

The term “determining”, as used herein, relates to the determination ofany parameter that can be useful in determining the evolution of apatient. As will be understood by those skilled in the art, theprediction of the outcome, although preferred to be, need not be correctfor 100% of the subjects to be diagnosed or evaluated. The term,however, requires that a statistically significant portion of subjectscan be identified as having an increased probability of having a givenoutcome. Whether a subject is statistically significant can bedetermined without further ado by the person skilled in the art usingvarious well known statistic evaluation tools, e.g., determination ofconfidence intervals, p-value determination, Student's t-test,Mann-Whitney test, etc. Details are found in Dowdy and Wearden,Statistics for Research, John Wiley & Sons, New York 1983. Preferredconfidence intervals are at least 50%, at least 60%, at least 70%, atleast 80%, at 90%, or at least 95%. The p-values are, preferably, 0.05,0.01, 0.005 or lower.

The term “prognosis”, as used herein, means the likelihood of recoveryfrom a disease or the prediction of the probable development or outcomeof a disease, including but not limited to predicting the length ofoverall survival (OS), 1 year survival (1YS), response to therapy (RT),disease-free survival, progression-free survival, and event-freesurvival. As will be understood by those skilled in the art, theprediction, although preferred to be, need not be correct for 100% ofthe subjects to be diagnosed or evaluated. The term, however, requiresthat a statistically significant portion of subjects can be identifiedas having an increased probability of having a given outcome. Whether asubject is statistically significant can be determined without furtherado by the person skilled in the art using various well known statisticevaluation tools, e.g., determination of confidence intervals, p-valuedetermination, Student's t-test, Mann-Whitney test, etc. Details arefound in Dowdy and Wearden, Statistics for Research, John Wiley & Sons,New York 1983. Preferred confidence intervals are at least 50%, at least60%, at least 70%, at least 80%, at least 90% at least 95%. The p-valuesare, preferably 0.05, 0.02, 0.01 or lower.

Standard criteria (Miller, et al. Cancer, 1981; 47(1): 207-14) can beused herewith to evaluate the clinical outcome of a patient in responseto a therapy. Any parameter which is widely accepted for determining theefficacy of treatments can be used for determining the clinical outcomeof a patient in response to a treatment and include, without limitation:

-   -   disease-free progression which, as used herein, describes the        proportion of subjects in complete remission who have had no        recurrence of disease during the time period under study.    -   disease-free survival (DFS), as used herewith, is understood as        the length of time after treatment for a disease during which a        subject survives with no sign of the disease.    -   objective response which, as used in the present invention,        describes the proportion of treated subjects in whom a complete        or partial response is observed.    -   tumor control which, as used in the present invention, relates        to the proportion of treated subjects in whom complete response,        partial response, minor response or stable disease ≥6 months is        observed.    -   progression free survival which, as used herein, is defined as        the time from start of treatment to the first measurement of        cancer growth.    -   Time to progression, as used herein, relates to the time after a        disease is treated until the disease starts to get worse. The        term “progression” has been previously defined.    -   six-month progression free survival which, as used herein,        relates to the percentage of subjects wherein free of        progression in the first six months after the initiation of the        therapy.    -   Overall survival (OS) which, as used herein, is defined as the        percentage of patients who survive after diagnosis of a primary        cancer.    -   median survival which, as used herein, relates to the time at        which half of the subjects enrolled in the study are still        alive, and    -   Cytogenetic risk stratification, which, as used herein, relates        to the probability of 5-year survival based on the chromosome        structure of the leukemic cells.

In a preferred embodiment, the clinical outcome of a patient is measuredby determining the cytogenetic risk stratification.

The first step of the in vitro method for determining the prognosis,comprises determining the expression level of type 1 5-HTR and/or type 25-HTR in cells of a sample from said subject selected from the groupconsisting of bone marrow, blood and lymph nodes. Determining the levelof expression of a biomarker, refers to determining the level ofexpression of a biomarker and/or the number of cells carrying abiomarker on its surface (i.e. a cell surface marker). Therein, thelevel of expression refers to the level of mRNA and/or the level ofprotein and/or the number of cells carrying a biomarker on its surface.Examples of methods for determining the expression level of a biomarkerhave been previously described.

In another preferred embodiment, the in vitro method for determining theprognosis of a subject suffering from a haematological malignancycomprises determining the expression level of type 1 5-HTR and/or type 25-HTR by measuring the level of mRNA encoded by the type 1 5-HTR genesand/or type 2 5-HTR genes, or of variants thereof. In another preferredembodiment, the expression level of type 1 5-HTR and/or type 2 5-HTR isdetermined by measuring the level of type 1 5-HTR proteins and/or type 25-HTR proteins, or of variants thereof. In a more preferred embodiment,the mRNA expression level is determined by PCR. In another preferredembodiment, the expression level of proteins or of variants thereof isdetermined by Western blot or immunocytochemistry. In a more preferredembodiment, the expression level of type 1 5-HTR and/or type 2 5-HTR isdetermined by semi-quantitative PCR.

In another preferred embodiment, the blood sample is peripheral blood.

In a preferred embodiment, the in vitro method for determining theprognosis of a subject suffering from a haematological malignancycomprises determining the level of type 1 5-HTR. In a more preferredembodiment, the type 1 5-HTR is selected from the group consisting of5-HTR 1A and 5-HTR-1B.

In a preferred embodiment, the method comprises determining theexpression level of 5-HTR 1A and 5-HTR-1B.

In a second step the in vitro method for determining the prognosiscomprises comparing the level of type 1 5-HTR and/or type 2 5-HTR with areference value. Said comparison allows concluding if the subject showsa good or a bad prognosis.

In a preferred embodiment, the prognosis determined by the in vitromethod of the invention is the probability of 5-year survival.

All the terms and embodiments previously described are equallyapplicable to this aspect of the invention.

6—Method for Monitoring the Effect of a Therapy

In another aspect, the invention relates to an in vitro method formonitoring the effect of a therapy in a subject suffering from ahaematological malignancy and being treated with said therapy whichcomprises:

-   -   a) determining the expression level of type 1 5-HTR and/or type        2 5-HTR in cells of a sample from said subject selected from the        group consisting of bone marrow, blood and lymph nodes, and    -   b) comparing said level with the expression level of type 1        5-HTR and/or type 2 5-HTR in cells of a sample from said subject        at an earlier point of time        wherein a decrease of the expression level of type 1 5-HTR        and/or type 2 5-HTR with respect to the level determined in a        sample from said subject at an earlier point of time is        indicative that the therapy is being effective or wherein an        increase of the expression level of type 1 5-HTR and/or type 2        5-HTR with respect to the level determined in a sample from said        subject at an earlier point of time is indicative that the        therapy is being ineffective.

In a preferred embodiment, the haematological malignancy is leukaemia,and more preferably acute myeloid leukaemia.

As a person skilled in the art understands, the therapy is directed totreating haematological malignancy. By way of non-limitative example acombination of cytarabine (ara-C) and daunorubicin (daunomycin) oridarubicin, fludarabine or topotecan can be used. In another embodimentthe therapy is a type 1 5-HTR inhibitor or a type 2 5-HTR inhibitor.

Methods for determining the expression level of type 1 5-HTR and/or type2 5-HTR have been previously described in detail.

In a preferred embodiment, the in vitro method for monitoring the effectof a therapy in a subject suffering from a haematological malignancycomprises determining the level of type 1 5-HTR. In a more preferredembodiment, the type 1 5-HTR is selected from the group consisting of5-HTR 1A and 5-HTR-1B.

In a preferred embodiment, the method comprises determining theexpression level of 5-HTR 1A and 5-HTR-1B.

In another preferred embodiment, the blood sample is peripheral blood.

In another preferred embodiment, the in vitro method for monitoring theeffect of a therapy in a subject suffering from a haematologicalmalignancy comprises determining the expression level of type 1 5-HTRand/or type 2 5-HTR by measuring the level of mRNA encoded by the type 15-HTR genes and/or type 2 5-HTR genes, or of variants thereof. Inanother preferred embodiment, the expression level of type 1 5-HTRand/or type 2 5-HTR is determined by measuring the level of type 1 5-HTRproteins and/or type 2 5-HTR proteins, or of variants thereof.

In a more preferred embodiment, the mRNA expression level is determinedby PCR. In another preferred embodiment, the expression level ofproteins or of variants thereof is determined by Western blot orimmunocytochemistry. In a more preferred embodiment, the expressionlevel of type 1 5-HTR and/or type 2 5-HTR is determined bysemi-quantitative PCR.

All the terms and embodiments previously described are equallyapplicable to this aspect of the invention.

7—Method for Designing a Customized Therapy

In another aspect, the invention relates to an in vitro method fordesigning a customized therapy for a subject diagnosed with ahaematological malignancy which comprises

-   -   a) determining the levels of the type 1 5-HTR and/or type 2        5-HTR-expressing cells in a sample from said subject selected        from the group consisting of bone marrow, blood and lymph nodes,        and    -   b) comparing said levels with a reference value        wherein increased levels of type 1 5-HTR and/or type 2        5-HTR-expressing cells with respect to the reference value are        indicative that the subject is to be treated with a type 1 5-HTR        inhibitor and/or a type 2 5-HTR inhibitor.

Designing a customized therapy to a subject diagnosed with ahaematological malignancy is understood as deciding, based on expressionof type 1 5-HTR and/or type 2 5-HTR, administering as appropriate a type1 5-HTR inhibitor and/or a type 2 5-HTR inhibitor.

In a preferred embodiment the 5-HTR inhibitor is selected from acompound of Table 1 or a pharmaceutically salt thereof.

In a more preferred embodiment, the 5-HTR inhibitor is selected from thegroup consisting of apomorphine methiothepin, amperozide and apharmaceutically acceptable salt thereof.

In a preferred embodiment, the in vitro method for designing acustomized therapy in a subject suffering from a haematologicalmalignancy comprises determining the expression level of type 1 5-HTR.In a more preferred embodiment, the type 1 5-HTR is selected from thegroup consisting of 5-HTR 1A and 5-HTR-1B.

In a preferred embodiment, the method comprises determining theexpression level of 5-HTR 1A and 5-HTR 1B.

In another preferred embodiment, the haematological malignancy isleukaemia, and more preferably acute myeloid leukaemia.

In another preferred embodiment, the blood sample is peripheral blood.

In another preferred embodiment, the in vitro method for monitoring theeffect of a therapy in a subject suffering from a haematologicalmalignancy comprises determining the expression level of type 1 5-HTRand/or type 2 5-HTR by measuring the level of mRNA encoded by the type 15-HTR genes and/or type 2 5-HTR genes, or of variants thereof. Inanother preferred embodiment, the expression level of type 1 5-HTRand/or type 2 5-HTR is determined by measuring the level of type 1 5-HTRproteins and/or type 2 5-HTR proteins, or of variants thereof. In a morepreferred embodiment, the mRNA expression level is determined by PCR. Inanother preferred embodiment, the expression level of proteins or ofvariants thereof is determined by Western blot or immunocytochemistry.In a more preferred embodiment, the expression level of type 1 5-HTRand/or type 2 5-HTR is determined by semi-quantitative PCR.

All the terms and embodiments previously described are equallyapplicable to this aspect of the invention.

The invention will be described by way of the following examples whichare to be considered as merely illustrative and not limitative of thescope of the invention.

EXAMPLES Material and Methods Example 1. Serotonin Receptor (5HTR)Antagonists Type 1 and 2 have a Cytotoxic Effect on AML Cell Lines

In order to test the anti-leukaemic effect of 5HTR antagonists, HL-60(Collins, Gallo et al. Nature. 1977; 270(5635):347-9), MonoMac-1(Steube, Teepe et al. Leuk Res. 1997; 21(4):327-35), KG-1 (Koeffler andGolde. Science. 1978; 200(4346):1153-4) and Kasumi-1 (Asou, Tashiro etal. Blood. 1991; 77(9):2031-6) AML (Acute Myeloid Leukaemia) cell lineswere treated with the broad 5HTR antagonist Apomorphine (CAS no.314-19-2) at different doses for 72 h. One million cells per mL wereseeded in complete RPMI media supplemented with 10% fetal bovine serumin a 96-well plate. The concentration used for the Apomorphine treatmentwas 0.1, 1 and 10 μM. As shown in FIG. 1, a reduction in cell viabilitywas detected in a dose-response fashion in all AML cell lines tested.

The human 5HTR family is composed by 7 subtypes (from 1 to 7) ofreceptors (Nichols and Nichols. Chem Rev 2008, 108:16414-41). HL-60,KG-1, MonoMac-1 and Kasumi-1 AML cell lines were incubated withsubtype-specific 5HTR antagonists: Apomorphine (type 1, 2 and 5),Methiothepin (type 1 and 2) (CAS no. 74611-28-2), Amperozide (type 2)(CAS no. 75558-90-6), GR113808 (type 4) (CAS no. 144625-51-4). 72 hafter treatment, cell viability was determined by flow cytometry. Cellswere stained with 7-AAD (CAS no. 7240-37-1) and Hoechst 33342 (CAS no.23491-52-3). Viability cells were identified by 7-AAD staining exclusionand the presence of Hoechst positive stain. Cell counts were measured byvolume. Only Apomorphine, Methiothepin and Amperozide induced cell deathon AML cell lines (FIG. 2); therefore, inhibition of 5THR type 1 and 2has cytotoxic effect on AML cells. In fact, the anti-leukaemic effectwas reversed in the presence of a competitive agonist such as thenatural ligand 5-HT (CAS no. 153-98-0) or 5-CT (CAS no. 74885-72-6).

Example 2. Serotonin Receptor (5HTR) Antagonists Type 1 and 2 have aCytotoxic Effect on Primary Patient AML Samples

Primary AML patient samples were treated with subtype 1 and 2 HTRantagonists for 24 and 72 h at 0.1, 1 and 10 μM. Five hundred thousandcells per mL were seeded in IMDM supplemented with insulin, transferrin,bovine serum albumin, IL3 and non-essential aminoacids. Cell viabilitywas measured by flow cytometry as described above. Similarly to AML celllines, both apomorphine and methiothepin treatment induced cell death onprimary AML blasts in a dose-response fashion (FIGS. 3 and 4).

Like the normal haematopoietic system, AML is thought to be organized asa hierarchy of distinct and functionally heterogeneous classes of cellsthat is ultimately sustained by a small number of leukaemic stem cells(LSCs) with enhanced self-renewal capacity, impaired differentiationability, and increase drug resistance (Bonnet and Dick Nat Med. 1997;3(7):730-7). The LSC population is found enriched inside theCD34+CD38-AML blast population. Apomorphine and methiothepin reducedcell viability of the most primitive LSC-enriched blast population(FIGS. 3 and 4) identified by flow cytometry based on the presence ofCD34 and absence of CD38. In fact, the reduction in the primitivefraction was significantly higher than in the bulk population; thus,5HTR antagonists selectively affect LSCs.

Example 3. 5HTR Antagonists Induced Differentiation on AML Cell Lines

HL-60, KG-1, MonoMac-1 and Kasumi-1 AML cell lines were treated withApomorphine and Methiothepin at different concentrations for 72 h asdescribed above. The expression of differentiation-associated surfacemarkers was measured by flow cytometry. In all AML cell lines, thepresence of 5HTR antagonists induced the expression of CD11c, CD11b andCD14 (FIG. 5).

Similarly, Apomorphine and Methiothepin induced the upregulation ofmyeloid markers on primary AML patient samples (FIGS. 6 and 7).

Example 4. 5HTR Antagonists have No Effect Neither on Healthy BloodCells Nor on Healthy Haematopoietic Stem/Progenitor Cells

Peripheral blood cells from healthy donors were isolated and treatedwith 5HTR antagonists using the same conditions as for primary patientAML samples. Unlike the latter, healthy blood cells remains unaffectedupon treatment with Apomorphine or Methiothepin (FIG. 8).

To study the effect of 5HTR antagonist treatment on haematopoieticstem/progenitor cells, mononuclear cells from umbilical cord bloodsamples from healthy donors were isolated and the lineage-negativefraction was cultured in the presence of Apomorphine. Unlike AML cells,primary haematopoietic stem/progenitor cells remain unaffected upontreatment. No significant changes in cell viability were observed in thefrequency of each population or the absolute number of cells (FIG. 9).

Example 5. 5HTR Antagonists Reduce the Clonogenic Capacity of AML Blastswhile Sparing Healthy Hematopoietic Stem Cells

Taking into account that 5HTR antagonists reduced the cell viability ofthe most primitive AML population, the clonogenic capacity upontreatment was investigated. Primary patient AML samples were cultured inthe presence of Apomorphine and Methiothepin for 18 h and plated in asemi-solid methylcellulose media for 14 days in the presence ofinstructive cytokines at a concentration of fifty thousand cells per mL.Colonies were counted by light microscopy based on morphology andcellularity. As shown in FIG. 10, both 5HTR antagonists reduced theclonogenic capacity of primary AML samples measured by the number ofCFU-B obtained.

Next, lineage-depleted umbilical cord blood cells were treated withApomorphine or Methiothepin as done above for AML cells. 14 days afterplating, colonies were counted by light microscopy based on morphologyand cellularity. None of the 5HTR antagonists affected the clonogeniccapacity of healthy haematopoietic stem/progenitor cells as measured byeither the total number of colonies or the frequency of each subtype ofCFU (FIG. 11).

Example 6. 5HTRs are Differentially Expressed on AML Patient Samples

To determine if 5HTRs are differentially expressed on AML patientsamples versus healthy blood samples, their expression on primarypatient AML peripheral blood samples, healthy donor peripheral bloodsamples and AML cell lines were studied by flow cytometry. As shown inFIG. 12, primary patient AML peripheral blood cells expressed 5HTRs in asignificant higher quantity compared to healthy donors.

Example 7. 5HTRs Expression Correlates with the Clinical Outcome in AMLPatients

5HTR1A and 5HTR1B mRNA expression levels were determined in primarypatient AML samples and correlated with the clinical outcome. Twopatient groups were defined based on the cytogenetic risk: good(favorable molecular group) and bad (unfavorable molecular group). Inboth cases, there was an association between high expression of each5HTR mRNA and worse clinical outcome (FIG. 13).

Example 8. Serotonin Receptor (5HTR) Antagonists Reduce the ClonogenicCapacity of AML Cells in Secondary CFUs

In order to assay the clonogenic capacity in vitro, equivalentapomorphine-, methiothepin- and vehicle-treated primary CFU-Bs from AMLpatients (as explained in example 5) were serially plated in asemi-solid methylcellulose media for 14 days in the presence ofinstructive cytokines at a concentration of fifty thousand cells per mLbut in the absence of any drug. Colonies were counted by lightmicroscopy based on morphology and cellularity. As shown in FIG. 14, areduction in the clonogenic capacity was observed upon treatment.

Example 9. Treatment with Serotonin Receptor Antagonists Reduced AMLBurden in an In Vivo Xenotransplantation Mouse Model, Sparing HealthyBlood Cells

Leukemia cells mainly reside in the bone marrow niche where the stromacell compartment provides paracrine signaling that strongly mediatestheir survival and proliferation and protects them from apoptosis ( ).Accordingly, conditioned immunodeficient NOD.Cg-Prkdc^(scid)Il2rg^(tm1Wjl)/SzJ (NSG) mice (Sanchez et al Leukemia. 2009 November;23(11):2109-17) were transplanted with human AML cells and left for 7days for the leukemia to be established. Afterwards, mice were treatedevery 2 days with a total of 4 intraperitoneal doses of apomorphine (5mg/kg weight) (Schmidt et al J Neurosci. 1982 March; 2(3):376-80) ormethiothepin (0.1 mg/kg weight) (Ginawi et al J Physiol Pharmacol. 2004June; 55(2):357-69) for 7 days. Both 5HTR1 antagonists produced asignificant reduction in AML burden in bone marrow (BM) as compared tovehicle-treated mice (FIG. 15).

Leukemia-initiating cells (LIC) or LSCs are responsible for theengraftment of AML cells in xenograft mouse models and are thought toinitiate and maintain the disease in humans. Primary AML patient sampleswere treated ex vivo with 5HTR antagonists for 18 h and transplantedinto conditioned immunodeficient NSG mice. Eight weeks aftertransplantation, murine bone marrow was analyzed for the presence ofhuman AML cells. As shown in FIG. 16, apomorphine- andmethiothepin-treated AML cells demonstrated less homing and engraftmentcapacity compared to vehicle-treated AML cells. Interestingly, anegligible effect was observed in the normal hematopoietic regenerationcapacity of lineage-depleted UCB cells after treatment with apomorphineand methiothepin (FIG. 16).

In order to assay the in vivo self-renewal capacity remaining in theengrafted samples, secondary transplants were performed. Less than 35%of AML cells were detected in mice injected with apomorphine- ormethiothepin-treated cells (FIG. 17). However, healthy hematopoieticstem cells retained their self-renewal and differentiation capacity upontreatment as shown by their regeneration potential in secondarytransplants (FIG. 17). Moreover, the clonogenic capacity of engraftedsamples was significantly reduced in AML cells treated with 5HTR1antagonists as measured by CFU assays. Interestingly, little effect isobserved in engrafted healthy HSCs (FIG. 17).

1-34. (canceled)
 35. A method of treating a subject suffering from acutemyeloid leukemia (AML) comprising determining the expression of type 1Bserotonin receptor (5-HTR 1B) in AML cells, wherein an administration ofa 5-HTR 1B inhibitor to the subject induces cell death in the AML cellsif the AML cells express 5-HTR 1B.
 36. The method of claim 35, whereinthe 5-HTR 1B inhibitor is selected from the group consisting ofmethiothepin, alprenolol, AR-A000002, asenapine, cynipindolol,GR-127,935, iodocyanopindolol, isamoltane, metergoline, oxprenolol,pindolol, propranolol, SN-216,641, yohimbine, GR-55562, SB-224289,SB-236057, and a pharmaceutically acceptable salt thereof.
 37. Themethod of claim 35, wherein the expression of 5-HTR 1B in AML cells isdetected by measuring the level of 5-HTR 1B proteins.
 38. The method ofclaim 35, wherein the expression of 5-HTR 1B in AML cells is detected bymeasuring the level of mRNA encoded by 5-HTR 1B genes.
 39. The method ofclaim 37, wherein the expression level of 5-HTR 1B proteins isdetermined by Western blot or immunocytochemistry.
 40. The method ofclaim 38, wherein the expression level of 5-HTR 1B genes is determinedby PCR.
 41. The method of claim 37, wherein the expression of 5-HTR 1Bis determined a blood sample.
 42. The method of claim 41, wherein theblood sample is a peripheral blood sample.
 43. A method of monitoringand treating a subject suffering from AML comprising: (a) administeringa 5-HTR 1B inhibitor to the subject, wherein the administration of the5-HTR 1B inhibitor to the subject induces cell death in the AML cells;and, (b) continuing administration of the 5-HTR 1B inhibitor to thesubject if the treatment with 5-HTR 1B inhibitor is being effective, or(c) discontinuing administration of the 5-HTR 1B inhibitor to thesubject if the treatment with 5-HTR 1B inhibitor is being ineffective,wherein (i) a decreased 5-HTR 1B expression level after the treatmentwith 5-HTR 1B inhibitor with respect to the 5-HTR 1B expression levelprior to the treatment with 5-HTR 1B inhibitor indicates that thetreatment with 5-HTR 1B inhibitor is effective; and, (ii) an increased5-HTR 1B expression level the treatment with 5-HTR 1B inhibitor withrespect to the 5-HTR 1B expression level prior to the treatment with5-HTR 1B inhibitor indicates that the treatment with 5-HTR 1B inhibitoris ineffective.
 44. The method of claim 43, wherein the 5-HTR 1Binhibitor is selected from the group consisting of methiothepin,alprenolol, AR-A000002, asenapine, cynipindolol, GR-127,935,iodocyanopindolol, isamoltane, metergoline, oxprenolol, pindolol,propranolol, SN-216,641, yohimbine, GR-55562, SB-224289, SB-236057, anda pharmaceutically acceptable salt thereof.
 45. The method of claim 43,wherein the expression of 5-HTR 1B in AML cells is detected by measuringthe level of 5-HTR 1B proteins.
 46. The method of claim 45, wherein theexpression of 5-HTR 1B in AML cells is detected by measuring the levelof mRNA encoded by 5-HTR 1B genes.
 47. The method of claim 45, whereinthe expression level of 5-HTR 1B proteins is determined by Western blotor immunocytochemistry.
 48. The method of claim 46, wherein theexpression level of 5-HTR 1B genes is determined by PCR.
 49. The methodof claim 43, wherein the expression of 5-HTR 1B is determined in a bloodsample.
 50. The method of claim 49, wherein the blood sample is aperipheral blood sample.